1. Field of Invention
The present invention provides HIV envelope immunogens that display multivalent epitopes. In particular, the immuogens are “webbed” HIV envelope immunogens in which native envelope structures are stabilized due to interactions with derivatives of M9 scorpion toxin.
2. Background of the Invention
Over 40 million people are infected worldwide with HIV-1, with the majority of cases in Africa (UNAIDS 2002). Although antiretroviral drugs can reduce viral replication and therefore slow immunopathogenesis, virtually all infected individuals progress to AIDS and ultimately death. Clearly, a vaccine is needed to control the spread of HIV.
It is likely that an effective HIV vaccine will consist of components that confer protection through CD8+ cytotoxic T lymphocytes (CTLs) and antibodies (Abs). CTLs can control replication of HIV and therefore slow pathogenesis, but do not prevent infection (see reviews in Letvin et al. Annu Rev. Immunol 20:73 (2002); McMichael and Hanke, Nature Rev. Immunol 2: 283(2002); Spearman 2003. Curr HIV Res 1:101 (2003)). Only Abs can completely prevent infection, resulting in so-called sterilizing immunity. Depending on the dosage, passive transfer of neutralizing monoclonal or polyclonal Abs against HIV-1 may confer sterile protection against challenge with pathogenic simian/human immunodeficiency chimeric virus in non-human primates (Mascola et al., J. Virol. 73: 4009 (1999); Mascola et al., Nat Med. 6:207 (2000); Parren et al., J. Virol. 75: 8340 (2001); Nishimura et al., Proc Natl Acad Sci USA. 100:15131 (2003)).
The exact mechanism by which antibodies confer protection in vivo or neutralize virus in vitro is unknown, but there is a positive correlation between protection and neutralization (Parren and Burton, Adv. Immunol 77:195(2001)). Neutralization probably occurs by the binding of Abs to a large number of sites on the virus, thus interfering with viral attachment to, and entry into host cells (Parren and Burton, Adv. Immunol 77:195(2001); Parren et al, J. Virol. 72: 3512 (1998); Burton, Nat. Rev. Immunol. 2:706 (2002)).
As alluded to above, the target of protective Abs to HIV is the 160 kDa envelope glycoprotein (Env). Env is synthesized as a precursor called gp160, which undergoes posttranslational modifications such as N-linked glycosylation and oligomerization in the endoplasmic reticulum (Earl et al., J. Virol. 65:2047 (1991)). Gp160 is cleaved by cellular proteases into gp120 and gp41 subunits in the Golgi apparatus (McCune et al, Cell 53: 55 (1988); Earl et al., J. Virol. 65: 2047(1991); Decroly et al, Febs Lett 405: 68 (1997); Hallenberger et al, J. Virol. 71: 1036 (1997)). Following cleavage, gp120 associates with gp41 through weak non-covalent interactions, and three gp120/gp41 dimers associate to form the mature Env trimer during transport to the cell surface (Kowalski et al, Science 237: 1351 (1987); Earl et al, J. Virol. 65:2047 (1991); Helseth et al., J. Virol 65: 2119 (1991)). The gp120 portion of the trimer is surface-exposed, whereas the gp41 portion of the trimer consists of a partially surface-exposed ectodomain, a transmembrane region, and a cytoplasmic tail that anchors the glycoprotein to the plasma membrane. Once Env trimers reach the surface of infected cells these molecules are incorporated into the budding virions through an interaction between the gp41 cytoplasmic domain and the capsid (Earl et al, Proc Natl Acad Sci USA. 87:648 (1990); Center et al, J Virol 76: 7863 (2002)).
During entry into a target cell, gp120 sequentially binds CD4 and chemokine receptors (such as CCR5 and CXCR4) on the target cell surface (Maddon et al, Cell 47: 333 (1986); Lifson et al., Nature. 323: 725 (1986); Dalgleish et al., Nature 312: 763 (1984); Klatzmann et al, Nature 312: 767 (1984); Deng et al., Nature 381: 661 (1996); Dragic et al, Nature 381: 667 (1996)). Receptor binding results in conformational changes that ultimately lead to fusion of viral and host cell membranes (a process believed to be mediated by the fusion peptide located at the N terminus of gp41) and, consequently, entry into the host cell.
Most Abs raised against envelope immunogens such as soluble monomeric gp120 or against the virus itself do not neutralize primary isolates of HIV-1 or protect the host from infection (Connor et al., J Virol 72: 1552 (1998); Mascola, J. Infect. Dis. 173: 340 (1996); Matthews, AIDS Res. Hum. Retroviruses 10: 631 (1994); Parren et al., AIDS 13 (Suppl. A):S137 (1999)). The molecular structure and antigenic properties of gp120 may in part explain the difficulty encountered in eliciting a neutralizing Ab response.
First, conserved epitopes that are located in parts of Env involved in cell entry, such as CD4- and coreceptor-binding sites are recessed within gp120 and poorly immunogenic (Kwong et al., Nature 393: 648 (1998); Wyatt et al., J Virol 69: 5723 (1995); Wyatt et al, Nature 393: 705 (1998)). Following CD4 binding, however, gp120 undergoes conformational changes that expose another class of conserved epitopes, namely CD4-induced (CD4i) epitopes, some of which are associated with the coreceptor-binding site in gp120 (Thali et al., J Virol 67: 3978 (1993); Wyatt et al., J Virol 69: 5723 (1995); Sullivan et al., J Virol 72: 4694 (1998)).
Second, the tertiary structure of the gp120 external surface has been divided into three functionally distinct domains: a neutralizing domain, a non-neutralizing domain, (Moore et al., J Virol 68: 469 (1994); Wyatt et al, Nature 393: 705 (1998)) and a silent domain (Wyatt et al, Nature 393: 705 (1998)). The non-neutralizing domain of gp120 is immunodominant in that it is highly immunogenic; however, Abs against this domain are non-neutralizing (Wyatt et al, Nature 393: 705 (1998)). Based on Ab-binding studies, it has been suggested that this domain is buried in the Env trimer but is exposed in monomeric gp120 and the uncleaved Env precursor, gp160 (Moore et al., J Virol 68: 469 (1994); Parren et al., Nat Med 3: 366 (1997); Wyatt et al, Nature 393: 705 (1998)).
Despite the difficulties discussed above, Ab-mediated protection against HIV is possible. Potent broadly neutralizing monoclonal antibodies (nmAbs) have been isolated from some HIV-positive patients that appear to be protected from progression to AIDS, or from experimental murine sources. The nmAbs include b12 (Burton et al., Science 266:1024 (1994)), X5 (Moulard et al., Proc Natl Acad Sci USA 99: 6913 (2002)), 2G12 (Trkola et al., J. Virol. 70: 1100 (1996)), m16 (Zhang et al., Antiviral Res 61: 161 (2004)), and m14 (Zhang et al., J Virol 78: 9233 (2004)) all of which recognize epitopes on gp120. A second subset of nmAbs, including 2F5 (Muster et al., J Virol 68: 4031 (1993)), Z13 (Zwick et al., J. Virol 75:10892 (2001)), and 4E10 (Stiegler et al, AIDS Res. Hum. Retroviruses 17: 1757 (2001)) binds to epitopes on gp41. Although the aforementioned nmAbs were raised against Env from clade B HIV-1 isolates, these nmAbs target conserved epitopes and each can neutralize isolates from other HIV-1clades in vitro, albeit with varying potencies (Zwick et al., J. Virol 75:10892 (2001); Stiegler et al., AIDS Res. Hum. Retroviruses 17: 1757 (2001); Trkola et al., J. Virol 69: 6609 (1995); Zhang et al., J Virol 78: 9233 (2004)). The challenge for HIV vaccine developers is to develop an immunogen that induces such Abs that display specificities similar to these broadly active nmAbs for a sustained period and therefore afford protection against an array of HIV-1 isolates.
Approaches to HIV Vaccine Design
During the early stages of HIV vaccine development, soluble monomeric gp120 was the most commonly used immunogen. However, it is now clear that gp120 elicits Abs that only neutralize HIV strains that express Env variants that were homologous to the gp120 or T cell line adapted (herein referred to as “TCLA”) isolates, that is, HIV isolates that have been cultured extensively in T cells. This latter phenomenon has been attributed to the fact that TCLA strains are significantly more susceptible to neutralization than HIV-1 isolates that are maintained on primary human PBMC (Daar et al., Proc Natl Acad Sci USA. 87: 6574 (1990); Moore et al., J. Virol. 69: 101 (1995). Given that these so called primary HIV-1 isolates are more representative of the infectious form of HIV-1, the inability of gp120 vaccines to induce Abs that neutralize primary isolates is probably the reason such vaccines were ineffective in Vaxgen's phase 3 clinical trials of bivalent recombinant gp120 (Francis et al., AIDS Res. Hum. Retroviruses 14 Suppl 3: S325 (1998); Lee et al., Vaccine 20: 563 (2001)).
Given this inadequacy, there is interest in modifying gp120 so as to alter the immunodominance pattern and enhance the immunogenicity of conserved neutralizing epitopes in this molecule (Pantophlet and Burton, Trends Mol Med. 9: 468 (2003). One strategy entails the use of selective mutagenesis resulting in glycosylation of residues in the non-neutralizing domain to redirect the immune response away from this immunodominant region (Pantophlet et al., J Virol 77: 5889 (2003); Pantophlet et al., J Virol 77: 642 (2003). Although early reports demonstrate that this strategy indeed diminishes the immunogenicity of glycosylated regions (Pantophlet et al., supra (2003), the usefulness of this strategy is dependent on the development of technologies that enhance the immunogenicity of conserved neutralizing epitopes. Otherwise, the result of this approach will be to simply reduce the immunogenicity of gp120 for humoral immunity. A solution to this paradox has yet to emerge in the prior art.
Another approach to HIV immunogen development entails the construction of immunogens derived from Env that form stable trimers. The rationale for this approach is to generate immunogens that mimic the structure of native Env trimers displayed on virions during transmission. Indeed, scanning transmission electron microscopy of virus-associated Env confirmed the existence of the trimeric tertiary structure (Center et al, J Virol 76: 7863 (2002)). Furthermore, Ab-binding studies suggest that neutralization of HIV-1 correlates with binding to native trimeric Env but not to monomeric gp120 (Parren et al., J Virol 72: 3512 (1998); Fouts et al., J Virol 71: 2779 (1997); Sullivan et al., J Virol 69: 4413 (1995)). Finally, a serendipitous advantage of this approach is that the highly immunogenic non-neutralizing domain of gp120 becomes occluded in the trimer form, which may quench the immunodominance of this domain and redirect the immune response to the neutralizing domain (Wyatt et al, Nature 393: 705 (1998)).
To construct stable trimeric Env immunogens, amino acids that enable the formation of gp41 trimers and gp120-gp41 interaction, which are necessary for the production of stable trimers, must be stabilized. Gp41 trimer formation has been enhanced by creating genetic fusions wherein the GCN4 trimer motifs, derived from a yeast transcription factor, or a T4 bacteriophage fibritin trimer motif were introduced into the carboxyl end of the ectodomain of gp41 (Yang et al., J Virol 74: 5716 (2000); Yang et al., J Virol 74: 4746 (2000); Yang et al., J Virol 76: 4634 (2002)). Antisera raised in mice (Yang et al., J Virol 75: 1165 (2001)) or macaques (Srivastava et al., J Virol 77: 11244 (2003)) against such trimeric Env immunogens neutralize heterologous primary isolates; however, the serum dilutions required to achieve 50 percent inhibition of HIV infectivity were usually between 1:40 and 1:20. Therefore, although this approach has some merit, immunogenicity of neutralizing epitopes in such trimeric Env immunogens thus far generated was poor and is considered to be too low to afford long-lived protection in human populations.
Stabilization of the gp120-gp41 interaction can be accomplished using one of two strategies. The first approach entails mutagenesis of the proteolytic cleavage site between gp120 and gp41; however, without additional modification proteolytically uncleaved Env tends to assemble into dimers and tetramers (Earl et al., Proc. Natl. Acad Sci USA 87: 648 (1990); Earl et al., J Virol 68: 3015 (1994); Earl et al., J Virol 71: 2674 (1997)). As an alternative, intersubunit disulphide bonds were introduced to stabilize the interaction between gp120 and gp41 (Binley et al., J Virol 74: 627 (2000); Yang et al., J Virol 74: 4746 (2000)).
A third strategy to modify gp120 entails exposing conserved CD4i epitopes in gp120. During HIV-1 entry into host cells, gp120 binds CD4 (Maddon et al., Cell. 47: 333 (1986); Lifson et al., Nature. 323: 725 (1986)) and undergoes conformational changes that expose conserved epitopes that are involved in coreceptor binding (such as CCR5 or CXCR4 chemokine receptors; Sattentau and Moore J Exp Med 174: 407 (1991); Jones et al., 1998 J Biol Chem 273: 404 (1998)). Evidence for the existence of CD4i epitopes was generated by studies showing that CD4-binding to gp120 enhances affinity of certain HIV-specific mAbs, including 17b and 48d, to the gp120-CD4 complex (Thali et al., J Virol 67: 3978 (1993)). Binding of mAbs to CD4i epitopes inhibits binding of the gp120-CD4 complex to CCR5, suggesting that such antibodies bind to or occlude the conserved coreceptor binding site, which is exposed only after gp120 binds to CD4 (Wu et al., Nature. 384: 179 (1996)). Indeed, structural studies suggest that the binding of gp120 to CD4 results in movement of the V1/V2 loops, thereby exposing the otherwise shielded coreceptor binding site (Wyatt et al., J Virol 69: 5723 (1995); Sullivan et al., J. Virol 72: 4694 (1998)).
Given the importance of CD4i epitopes in HIV infectivity, one approach to inducing broadly nmAbs to HIV is to create gp120 immunogens that surface-expose and lock-in-place CD4i epitopes (Pal et al., Virology 194:833 (1993); DeVico et al., Virology 218: 258 (1996); Hone et al., J Hum Virol 5: 17 (2002); Fouts et al., FEMS Immuno Med Microbiol 37: 129 (2003)). Indeed, gp120 or gp140 chemically cross-linked to soluble human CD4 (shCD4), or genetic fusions of gp120 and the D1D2 domains of human CD4induce antibodies in mice, goats and macaques that neutralize primary HIV-1 isolates across clades and regardless of coreceptor usage (DeVico et al., Virology 218: 258 (1996); Fouts et al., Proc Natl Acad Sci USA 99: 11842 (2002); Onyabe et al, unpublished)). It is worth noting that in both the goat and macaque studies, the source of gp120 in the complex was a TCLA strain (HIV-1IIIB), yet it elicited Ab that neutralized primary isolates.
Despite the encouraging observations above, concern over the use of human CD4 in a vaccine will hinder development of such immunogens beyond the laboratory. To circumvent the regulatory problems associated with the use of CD4, a CD4 mimetic, called M9 (Vita et al., Proc Natl Acad Sci USA 96: 13091 (1999), has the potential to be used in such immunogens. M9 is derived from scorpion toxin and competes with CD4 for binding to gp120 (Vita et al., Proc Natl Acad Sci USA 96: 13091 (1999); Zhang et al., Biochemistry. 38: 9405 (1999); Martin et al., Nat Biotech 21: 71 (2003)). More importantly, M9 and analog derivatives of this molecule induce conformational changes that expose CD4i epitopes (Vita et al., Proc Natl Acad Sci USA 96: 13091 (1999); Zhang et al., Biochemistry. 38: 9405 (1999); Martin et al., Nat Biotech 21: 71 (2003)), prevent cell-cell fusion of mammalian cells expressing HIV-1 envelope, and neutralize infectivity of HIV-1 strains in vitro regardless of coreceptor usage (Vita et al., Proc Natl Acad Sci USA 96: 13091 (1999); Martin et al., Nat Biotech 21: 71 (2003)). Unfortunately, a recent report indicated that gp120-M9 fusion protein failed to induce neutralizing antibodies to primary HIV isolates (Varadarajan et al. J. Virol. 79: 1713 (2005)).
It is clear from the foregoing discussion that the HIV Env immunogens currently available do not elicit high-titer broadly neutralizing antibodies to HIV. Therefore, there continues to be a need to develop HIV Env immunogens that induce antibodies that display broad specificity to native epitopes on HIV virions and/or possess broadly neutralizing activity against a wide array of primary HIV isolates.